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Investigating the aetiology of respiratory tract infections in children admitted to Tygerberg Children’s Hospital using molecular methods and viral culture

Maree, Leana (2012-12)

Thesis (MMed)--Stellenbosch University, 2012.

Includes bibliography

Thesis

ENGLISH ABSTRACT: Introduction
Acute respiratory tract infections cause significant morbidity and mortality worldwide, and are
the main reason for the utilisation of health care services. Identifying the aetiological cause of lower
respiratory tract infections (LRTIs) is difficult at the best of times, and more than 20 viruses and bacteria
have been associated with LRTIs, which cannot be distinguished with clinical examination alone. Viruses
can be detected in respiratory samples by a variety of methods, and without exception molecular
methods have proven to be more sensitive than non-molecular-based tests. The increased sensitivity of
molecular methods may assist in expanding our knowledge of the pathogenesis of severe respiratory
tract infections, and could have a positive influence on patient management, infection control,
vaccination strategies and public health.
Aims and objectives
1. Determine the viral causes of lower respiratory tract infections requiring admission in using shell
vial culture with immunofluorescent staining and two multiplex PCR assays, the Seeplex® RV15
ACE Detection system (Seeplex® RV15 ACE) and the Respiratory Multiplex Real-Time RT-PCR
LightMix® Customised Kit (Resp Multiplex RT-PCR).
2. Compare the Seeplex® RV15 ACE and the Resp Multiplex RT-PCR with shell vial culture for the
detection of respiratory viruses in routine diagnostic respiratory samples.
3. Examine the demographic and clinical characteristics associated with each respiratory viral
pathogen.
Materials and Methods
One hundred and thirty-eight paediatric patients, admitted to Tygerberg Children’s Hospital from
May 2010 to August 2010 with a presumptive diagnosis of an acute respiratory tract infection were
included in the study. Nasopharyngeal or tracheal aspirates were collected, and all samples were tested
by all three diagnostic methods. Clinical, demographic and laboratory data were collected through a
systematic review of medical and laboratory records and subsequently anonymised
Results
Thirty-seven viruses were detected in 36 samples (26.1%) by shell vial culture with
immunofluorescent staining; 169 viruses in 102 samples (73.9%) with the Seeplex® RV15 ACE; and 90
viruses in 73 samples (52.9%) with the Resp Multiplex RT-PCR. Shell vial culture had excellent specificity,
but low sensitivity for all of the respiratory viruses. Conversely, the Seeplex® RV15 ACE had excellent
sensitivity for all viruses, but slightly lower specificity. This was due to the detection of additional viruses,
which may have been true positives due to the increased sensitivity of this assay. The Resp Multiplex RTPCR
had excellent sensitivity and specificity.
At least one respiratory pathogen could be identified in 80% of the patients. At least one virus
was detected in 57% of patients, bacterial micro-organisms in 6%, and both viral and bacterial pathogens
in 17%. Viral-bacterial co-infections were associated with increased severity compared to other
infections, as these children were more likely to receive steroids and a blood transfusion (p = 0.002), and
more likely to require mechanical ventilation (p < 0.001) and admission to the intensive care unit (p =
0.04).
Conclusions
We confirmed that molecular techniques are significantly more sensitive than shell vial culture
for the detection of respiratory viruses in children. Due to their highly specific nature and the genetic
variability observed in viruses, an excellent, continuous quality control programme is essential to ensure
the continued superiority of these assays. Viral-bacterial co-infection is associated with increased
severity of LRTIs in children. Further research is needed to elucidate the precise pathogenic and
immunologic mechanism of this interaction.